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Strain & Vector for use

Strain & Vector for use. Wei ZHENG 8th, Jul, 2008. Lambda InCh a simple E.Coli plasmid-chromosome shuttle system. Advantages. No risk of >1 copy of target gene in our machine Stable integration of a single copy of the plasmid fragment

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Strain & Vector for use

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  1. Strain & Vector for use Wei ZHENG 8th, Jul, 2008

  2. Lambda InCha simple E.Coli plasmid-chromosome shuttle system

  3. Advantages • No risk of >1 copy of target gene in our machine • Stable integration of a single copy of the plasmid fragment • Most ordinary E.Coli strains and a variety of pBR322-derived Amp-resistant plasmid can be used • λphage as the only specialized vector required

  4. General principle • Recombination of the desired plasmid-encoded genes onto lambda InCh • Integration of the recombinant lambda InCh into the chromosome at the lambda attachment site • Deletion of most of the lambda genes, including the att site from the chromosome of the lysogen

  5. Two homologous region • “Near ori” – at the region near pBR322 replicative origin • A fragment of the bla gene of pBR322

  6. pBR322

  7. pUC18

  8. How to select the lysogens? • Low frequency transducing (LFT) lysate: - typically10-3 of the population are recombinant phages, having complete bla gene (Ampr) • Heat-inducible cI857 repressor: - selection of the temperature- independent derivatives at 42oC

  9. E.Coli Strains

  10. E.Coli strain • BBa_V1001 DH5a • Genotype F-φ80dlacZΔM15 Δ(lacZYA-argF)U169 deoR, recA1 endA1 hsdR17(rk- mk+ phoA supE44 λ- thi-1 gyrA96 relA1

  11. Strain: BBa_V1001 DH5a • Vector: -λInCh2 - pUC18

  12. More Information • Gallery of λInCh Homology Maps http://beck2.med.harvard.edu/resources/InCh/lambda_InCh_frames.htm • λInCh page http://beck2.med.harvard.edu/resources/InCh/lambda_InCh_frames.htm • Manual

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